The infectious agent responsible for acquired immunodeficiency syndrome (AIDS) and its prodromal phases, AIDS-related complex (ARC) and lymphadenopathy syndrome (LAS), is a novel lymphotrophic retrovirus. The virus has been variously termed LAV, HTLV-III, ARV, and most recently HIV.
As the spread of HIV reaches pandemic proportions, the treatment of infected individuals and preventing the transmission to uninfected individuals at risk of exposure is of paramount concern. A variety of therapeutic strategies have targeted different stages in the life cycle of the virus and are outlined in Mitsuya and Broder, 1987, Nature 325:773. One approach involves the use of antibodies which bind to the virus and inhibit viral replication, either by interfering with viral entry into host cells or by some other mechanism. Once the viral component(s) susceptible to antibody intervention are identified, it is hoped that antibody titers sufficient to neutralize the infectivity of the virus could be engendered by vaccination or, alternatively, by the passive administration of immune globulins or monoclonal antibodies of the desired specificity.
The envelope glycoproteins of most retroviruses are thought to react with receptor molecules on the surface of susceptible cells, thereby determining (the virus' infectivity for certain hosts. Antibodies that bind to the glycoproteins may block the interaction of the virus with the cell receptors, neutralizing the infectivity of the virus. See generally, The Molecular Biology of Tumor Viruses, 534 (J. Tooze, ed., 1973) and RNA Tumor Viruses, 226, 236 (R. Weiss et al., eds., 1982), both of which are incorporated herein in their entirety by reference. See also, Gonzalez-Scarano et al., 1982, Virology 120:42 (La Crosse Virus); Matsuno and Inouye, 1983, Infect. Immun. 39:155 (Neonatal Calf Diarrhea Virus); and Mathews et al., 1982, J. Immunol., 129:2763 (Encephalomyelitis Virus).
The general structure of HIV is that of a ribonucleoprotein core surrounded by a lipid-containing envelope which the virus acquires during the course of budding from the membrane of the infected host cell. Embedded within the envelope and projecting outward are the viral encoded glycoproteins. The envelope glycoproteins of HIV are initially synthesized in the infected cell as a precursor molecule of 150,000-160,000 daltons (gp150 or gp160), which is then processed in the cell into an N-terminal fragment of 110,000-120,000 daltons (gp110 or gp120) to generate the external glycoprotein, and a C-terminal fragment of 41,000-46,000 daltons (gp41), which represents the transmembrane envelope glycoprotein.
For the reasons discussed above, the gp110 glycoprotein of HIV has been the object of much investigation as a potential target for interrupting the virus' life cycle. Sera from HIV infected individuals have been shown to neutralize HIV in vitro, and antibodies that bind to purified gp110 are present in the sera Robert-Guroff et al., 1985, Nature 316:72; Weiss et al., 1985, Nature 316:69; and Mathews et al., 1986, Proc. Natl. Acad. Sci. U.S.A., 83:9709. Purified and recombinant gp110 stimulated the production of neutralizing serum antibodies when used to immunize animals, Robey et al., 1986, Proc. Natl. Acad. Sci. U.S.A., 83:7023; Lasky et al., 1986, Science 233:209; and a human, Zagury et al., 1986, Nature 326:249. Binding of the gp110 molecule to the CD4 (T4) receptor has also been shown, and monoclonal antibodies which recognize certain epitopes of the CD4 receptor have been shown to block HIV binding, syncytia formation and infectivity. McDougal et al., 1986, Science 231:382. Putney et al. (1986, Science 234:1392) elicited neutralizing serum antibodies in animals after immunizing with a recombinant fusion protein containing the carboxyl-terminal half of the gp110 molecule and further demonstrated that glycosylation of the envelope protein is unnecessary for a neutralizing antibody response.
A subunit vaccine for AIDS utilizing the HIV gp110 molecule or portions thereof may thus be desirable. Subunit vaccines are an alternative to vaccines prepared from inactivated or attenuated viruses. Inactivated vaccines are worrisome due to the possible failure to kill all of the viral particles, and attenuated viruses may possess the ability to mutate and regain their disease-causing capability. With subunit vaccines, only those portions of the virus that contain the antigens or epitopes that are capable of eliciting immune responses, i.e., neutralizing antibodies, ADCC, and cytotoxic T-cell response, are used to immunize the host. A major advantage of subunit vaccines is that irrelevant viral material is excluded.
Viral subunits for use in a vaccine can be generated by several methods. By way of example, the envelope glycoprotein can be expressed and purified from a bacterial host, although this molecule would lack most post-translational modifications (such as glycosylation) or other processing. Such modification may be obtained using a eukaryotic expression system, such as yeast or cultured mammalian cells. Viral genes have been introduced into mammalian cells using the vaccinia virus as a vector. See, for example, Mackett, M., et al., 1982, Proc. Nat. Acad. Sci. U.S.A. 79:7415; Panicali, D. and Paoletti, E., 1982, Proc. Nat. Acad. Sci. U.S.A. 79:4927. Recombinant vaccinia virus may be constructed according to the method of Hu et al., Nature 320:537 (1986) or Chakrabarti et al., Nature 320:535 (1986), both of which are incorporated herein by reference. In these systems viral glycoproteins produced by cells infected with recombinant vaccinia are appropriately glycosylated and may be transported to the cell surface for extrusion and ultimate isolation.
An important step in the production of a subunit vaccine is adequate purification of the desired glycoprotein from the complex mixture of the expression system. Several methods can be used to accomplish the purification. These include but are not limited to preparative polyacrylamide gel electrophoresis, gel permeation chromatography and various methods of chromatography (i.e., ion exchange, reverse phase, immunoaffinity, hydrophobic interaction) and others. Most of these methods are used in various combinations to achieve substantially pure preparations (Kleid, D. G., et al., 1981, Science 214:1125; Cabradilla, C. D., et al., 1986, Biotechnology 4:128, Dowbenko, D. J., 1985, Proc. Nat. Acad. Sci. U.S.A. 82:7748) which are incorporated herein by reference.
Methods which would reduce the number of steps required to achieve maximum purification of a particular viral antigen from a complex expression mixture are needed to manufacture subunit vaccines. The efficient separation of the antigens from extraneous components could be accomplished using immunoaffinity chromatography. This technique, also known as immunoadsorption, consists in principle of the selective adsorption of an antigen to a solid support on which a specific antibody has been covalently attached. The selectively adsorbed antigen is then eluted from such an antibody affinity adsorbent by changing, for example, the pH and/or ionic strength of the buffer.
Polyclonal antibodies, obtained from animals immunized with the desired antigen or from naturally infected individuals (see, for example, Lasky et al., supra), have frequently been used as immunoadsorbants, but, in general these reagents present substantial disadvantages, such as (i) not all of the antibodies bound to the insoluble support are specific for the molecule of interest, necessitating additional purification; (ii) yields of the desired antigen are frequently low; and (iii) antibody affinities often vary from one preparation to another, requiring modifications in elution procedures. The use of monoclonal antibodies specific for the desired viral antigen to be used in the subunit vaccine preparation, rather than polyclonal antibodies, would circumvent these difficulties.
Murine monoclonal antibodies that bind HIV antigens have been described. Several groups have reported monoclonal antibodies specific for the core protein p25 (see, for example, di Marzo Veronese, et al., 1985, Proc. Nat. Acad. Sci. U.S.A. 82:5199 and Chassagne, J., et al., 1986, J. Immunol. 136:1442). Monoclonal antibodies specific for the membrane glycoprotein gp41 have also been reported (see, for example, di Marzo Veronese, et al. 1985, Science 229:1402).
There remains a need in the art for monoclonal antibodies specific for epitopes within well defined regions of the major envelope glycoprotein, gp110. Monoclonal antibodies which bind these regions and cause a reduction in or elimination of the replication and transmissibility of HIV would have substantial therapeutic and prophylactic utility. Moreover, the monoclonal antibodies could also be used to purify the desired region of gp110 from disrupted virus or recombinant expression systems for use in vaccines, for example. Additionally, the region containing the epitope(s) recognized by the monoclonal antibodies could be chemically synthesized, thereby avoiding the difficulties inherent in purification and administration of larger fragments of the gp110 molecule. The present invention fulfills these and other related needs.